Prostate cancer is a highly prevalent problem in the VA health care system. There are few therapies for men with cancer outside of the prostate; these are the men most in need of treatment because they are at the highest risk of death from the disease. This project will develop a vaccine therapy for prostate cancer using recombinant cytomegalovirus (CMV). CMV vectors are fundamentally different kind of vaccine, not just another vector because a) they are the only vector system providing persistent immune stimulation, b) anti-vector immunity is irrelevant, c) they induce a much broader T cell response directed against unconventional epitopes, d) they work even if they are spread-deficient. Finally, this is the first vector system that was able to induce an immune response that was able to control, even clear, highly virulent Simian Immunodeficiency Virus (SIV). Nothing else ever tried against this SIV has shown any similar efficacy. Recombinant CMV containing Human Immunodeficiency Virus sequences are in clinical development with spread-deficient HCMV/HIV vectors and expect to be in phase I clinical trials in 2016 in healthy adults. This proposal will explore the use of CMV-based viral vectors expressing various forms of Prostate Specific Antigen (PSA) as a therapy for prostate cancer in mice. It is clear that PSA is an antigen; it is a dominant antigen in men with autoimmune prostatitis where the immune system attacks the normal prostate. The study will utilize Transgenic Mouse Adenocarcinoma of the Prostate (TRAMP) cancer cells engineered to express PSA (TRAMP-PSA). A humanized mouse model of prostate cancer has been developed using TRAMP-PSA cancer cells. CMV has strong species specificity; the experiments require murine CMV (mCMV). Preliminary data showed that a murine CMV viral construct expressing PSA induced a protective anti-tumor immune response in mice challenged with TRAMP-PSA showing promise for the approach. Human CMV causes life-long infection in humans and is highly prevalent. More than half of the U.S population has been infected with CMV. While CMV rarely causes disease in healthy individuals, the virus can cause disease in immunosuppressed patients. The virus also has the potential of spreading into the environment from vaccinated patients. These characteristics present substantial hurdles to translation of a CMV-PSA vaccine into clinical use. Recent findings from several laboratories suggested that complete virus replication may not be necessary for induction and maintenance of the strong immune response against CMV. The virus can be readily engineered to be fully infectious but unable to make progeny. Such spread- deficient vectors are much more translatable to human use. The goal of current proposal is to determine whether the level of immunity induced by a spread- deficient version of the mCMV vector encoding PSA is sufficient to maintain the anti-tumor effect observed for its wild-type mCMV counterpart. The study will also determine whether the mCMV-PSA vaccine is an effective treatment for mice spontaneously developing the TRAMP tumor by starting treatment in the animals when their tumor has started to develop more closely modeling human prostate cancer. Finally, CMV will be tested in combination with the immune checkpoint inhibitor anti-CTLA4 to assess augmentation of the anti- tumor effect. The study consists of four aims: 1) To test the mCMV-PSA vectors in combination with anti- CTLA-4, 2) To test the CMV-PSA vectors in the spontaneous TRAMP mouse model more closely mimicking the human disease, 3) To characterize phenotype and function of PSA-specific CD8 and CD4 T cells induced by mCMV-PSA and, 4) To construct a spread-deficient version of mCMV-PSA and test its anti-tumor effect in transgenic mouse models of prostate cancer. The study will produce the final pre-clinical data needed to move forward with the creation of a promising therapy for prostate cancer in men.